The instant invention pertains to polymers. In particular, the instant invention pertains to polymers, which are useful in capillary electrophoresis. Such polymers may be used as dynamic coatings for the inner surfaces of capillaries used for capillary electrophoresis and are also useful as media contained within such capillaries. The instant invention further pertains to a method for making such polymers and to a capillary containing such polymeric coatings and/or media.
During the last decade, capillary electrophoresis (CE) has developed into a powerful analytical method due to its flexibility and low volume requirement. CE provides fast and efficient separations and offers the following advantages over conventional slab gel electrophoresis:
a) heat dissipation is very efficient in CE and Joule heating is minimized. This ensures negligible temperature gradients and thus reduces peak broadening. Due to these effects, strong electric fields (typically up to 400 V/cm) can be used, therefore reducing run time and diffusion, which again leads to smaller peak widths;
b) CE is compatible with a variety of detection methods, such as absorption, laser induced fluorescence (LIF), mass spectrometry, chemiluminescence, voltammetry etc. In the case of DNA separation, CE also offers full compatibility to existing biochemistry.
c) CE systems can inject directly from a variety of sample formats (e.g., Eppendorf tubes, microtitre plates, etc.) and even from single cells;
d) the separation process can be fully automated. Separation matrices and samples can be automatically injected, therefore avoiding the time consuming procedure of gel casting and sample loading;
e) multi-capillary devices offer the possibility to analyze samples in parallel.
Despite these positive features, room for improvement continues to exist in the area of CE. Improving the reproducibility of analysis times is of particular importance. Analyte effective mobility (xcexceff) in CE is a constant value depending on buffer composition and temperature. On the other hand, apparent mobility is often less reproducible, as electroosmotic mobility (xcexceo) can vary from run-to-run in a more a unpredictable way. Interaction of the analyte with the inner surface of the capillary contributes significantly to electroosmotic flow (EOF) variation. A number of CE applications, such as those involving the analysis of DNA and SDS-protein complexes, benefit from reduced EOF. For example, in the analysis of DNA, EOF suppression is required to prevent migration of the separation matrix out of the capillary and to avoid adsorption of protein contaminants and dyes to the capillary surface.
Several methods have been developed to control the surface properties of silica capillaries. Such methods include using a background electrolyte with suitable characteristics, dynamically coating the capillary surface with polymeric additives contained in the running buffer, and coating the capillary surface through covalent silanols derivatization. Although a number of different dynamic coatings produced by adsorption of a polymer from an aqueous solution have been described, a common problem of these coatings is that the polymer can be easily removed from the capillary wall simply by washing with water. Therefore, unless otherwise stabilized, these coating are efficient in suppressing EOF only when a small amount of polymer is dissolved in the running buffer and can replace the polymer removed by the water from the surface.
The difficulties associated with performing chemical derivatization on silica micro-channels represent a significant obstacle to the development of innovative techniques such as micro-chip technology. Multiple capillary array technology, where as much as 96 capillaries must be handled at a same time, requires very simple and reliable coating procedures. Accordingly, there exists a strong scientific and industrial interest in coating procedures that do not require the use of organic solvents, high viscous solutions, and elevated temperatures.
The instant invention relates to the use of uncharged water-soluble polymers to suppress electroosmotic flow in capillary electrophoresis. The subject polymers adsorb onto the capillary surface, forming a highly hydrophilic, dynamic coating which achieves EOF suppression without the addition of any polymer to the running buffer. The coatings of the invention possess an affinity for the capillary surface that may last for approximately 20 hours of continuous use, under an electric field in a running buffer containing 8M urea, at a pH 8.5 at 45xc2x0 C. The subject polymers are also useful as separation media contained within the capillary column. The instant coatings and media are especially suitable for applications pertaining to the electrophoretic separation of various biomolecules, such as protein and DNA.
The polymers useful in the instant invention are copolymers of various derivatives of acrylamide and methacrylamide monomers with various glycidyl group containing monomers e.g.,. dimethylacrylamide and allyl glycidyl ether-epoxy poly(DMA)-, copolymers of various derivatives of acrylamide and methacrylamide with various allyl group containing carbohydrates and various glycidyl group containing monomers, such as allyl xcex2-D-pyranoside (typically xcex2-D-glucopyranoside) or allyl xcex2-D-furanoside allyl glycidyl ether-epoxy poly(AG-AA) and copolymers of four different monomers including various acryl and methacrylamide, various allyl group containing carbohydrates, various glycidyl group containing monomer and various diol group containing monomer, such as acrylamide, allyl xcex2-D-pyranoside (typically xcex2-D-galactopyranoside or N-allylgluconamide) or allyl xcex2-D-furanoside, allyl glycydyl ether and allyoxy-1,2 propanediol-epoxy poly(AGal-AA-APD).
The instant invention also pertains to a capillary coated and/or filled with the above polymers and to a method for separating biomolecules using such a capillary.